Comparisons of subunit 5A and 5B isoenzymes of yeast cytochrome c oxidase.
Comparisons of subunit 5A and 5B isoenzymes of yeast cytochrome c oxidase.
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DOI：
10.1042/bj20140732
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发表时间：
2014-12-15
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通讯作者：
Rich PR
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作者：
Dodia R;Meunier B;Kay CW;Rich PR
酿酒酵母细胞色素c氧化酶（CcO）的亚基5是装配所必需的，有两种亚型，5A和5 B。5A在常氧条件下表示，而5 B在非常低的氧张力下表示。因此，COX 5A缺失的菌株（Δ cox 5A）在常氧条件下没有或只有低水平的CcO，使其呼吸缺陷。先前的研究已经报道，呼吸生长可以通过将Δ cox 5A与编码COX 5 B表达阻遏物的ROX 1突变相结合来恢复。在这些突变体中，5 B同工酶表达水平为野生型（5A同工酶）的30-50%，并表现出最大催化活性，比5A同工酶快3倍。为了研究这种效应的起源，我们构建了一种突变株，其中COX 5 B取代COX 5A启动子下游的COX 5A。该菌株表达野生型水平的5 B同工酶，而没有由ROX 1突变引起的额外影响的并发症。当以这种方式产生时，同工酶在它们的最大催化活性或它们对氧或细胞色素c的亲和力方面没有显着差异。因此，在rox 1突变体的5 B同工酶的活性升高不是简单地通过交换异构体，必须从一个额外的效果，仍然有待解决。具有亚基5A或5 B的酵母细胞色素c氧化酶同工酶可表现出不同的动力学性质。我们证明，这些差异不是由简单的5A/5 B替换诱导的，并且该效果还需要表达水平的变化和/或未定义的翻译后效应。
Subunit 5 of Saccharomyces cerevisiae cytochrome c oxidase (CcO) is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions, whereas 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory deficient. Previous studies have reported that respiratory growth could be restored by combining Δcox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants, 5B isoenzyme expression level was 30–50% of wild-type (5A isoenzyme) and exhibited a maximum catalytic activity up to 3-fold faster than that of 5A isoenzyme. To investigate the origin of this effect, we constructed a mutant strain in which COX5B replaced COX5A downstream of the COX5A promoter. This strain expressed wild-type levels of the 5B isoenzyme, without the complication of additional effects caused by mutation of ROX1. When produced this way, the isoenzymes displayed no significant differences in their maximum catalytic activities or in their affinities for oxygen or cytochrome c. Hence the elevated activity of the 5B isoenzyme in the rox1 mutant is not caused simply by exchange of isoforms and must arise from an additional effect that remains to be resolved. Yeast cytochrome c oxidase isoenzymes with subunits 5A or 5B can exhibit different kinetic properties. We demonstrate that these differences are not induced by simple 5A/5B replacement and that the effect additionally requires expression level changes and/or undefined post-translational effects.